ABSTRACT Angiogenic vessels form sprouts that migrate outward and interconnect with other vessels or sprouts to form a vessel network. Sprouts eventually are guided by cues such as VEGF from distant target sites, but it is not clear how sprouts initially move away from the parent vessel. Moreover, vessel networks often form in the absence of a strong VEGF gradient, yet sprouts still migrate outward from the parent vessel. An emerging concept is that angiogenic vessels are heterogeneous for endothelial phenotypes; for example both tip cells and stalk cells are required to form a sprout. We propose that endothelial cells adjacent to newly forming sprouts are also heterogeneous, and that the sprout phenotype is elaborated with input from cells in these lateral base areas. Thus we propose a unique role for the parent vessel in local sprout guidance. Preliminary data supports our central hypothesis that forming sprouts and their filopodia sensors are guided outward by cues from endothelial cells adjacent to the sprout in the vessel, and that Flt-1 (VEGFR-1) has a critical role in this local sprout guidance. Loss of this guidance mechanism leads to vessel dysmorphogenesis and perturbed vessel function. In this renewal application we will determine the molecular requirements for this novel local sprout guidance, and we will determine how endothelial cells cross-talk in developing vessels to integrate signals from the Notch pathway and the VEGF signaling pathway to set up localized and distinct endothelial cell phenotypes. We present three aims organized to elucidate molecular and cellular mechanisms responsible for this guidance, and experiments that will critically test the role of Flt-1 and Notch signaling in this process, both in fixed samples and via dynamic image analysis. We will take advantage of the strengths of a developmental model using stem cell-derived blood vessels, and we will complement these experiments with in vivo experiments in the yolk sac and the retina. This information is important to our ability to reconstitute a vascular plexus for therapeutic purposes. )